The present invention relates to a method of producing in a high yield and in a large amount cells of bacteria of the genus Pseudomonas having a high nitrile hydratase activity.
In recent years, there have been increasing attempts to utilize microorganisms and enzymes as they are or in immobilized state as catalysts for various single or complex chemical reactions.
Nitrile hydratase is known as an enzyme capable of hydrating nitriles to produce the corresponding amides. (Reference: Agric. Biol. Chem. 46 1165(1982)) As one example of the utilization of this enzyme, a method for preparation of amides from nitriles having 2 to 4 carbon atoms in the presence of bacteria having nitrile hydratase has been proposed. (References: Japanese Patent Pub. No. 37951/1984, U.S. Pat. No. 4,637,982 and Agric. Biol. Chem. 46 1183(1982))
Further, several methods for cultivation of such bacteria have been proposed. (Japanese Patent Pub. No. 43996/1986, No. 43997/1986, No. 43998/1986, No. 43999/1986, EP 0109083, U.S. Pat. No. 4,661,457, No. 4,661,456 and No. 4,555,487)
In order to utilize bacteria having nitrile hydratase activity on a commercial scale, it is necessary to produce bacteria in a large amount and hence to scale up a cultivation tank suitably from an experimental tank of the beaker scale.
The bacteria of the genus Pseudomonas capable of producing nitrile hydratase must be cultivated under aerobic conditions (at an oxygen feed rate of about 3.5 kg-O.sub.2 /m.sup.3 .multidot.hr.), for example, by agitation under aeration for proliferation of cells and expression of the activity thereof. When these bacteria were cultivated in a large-volume, i.e., scaled up, cultivation tank, however, both the cell concentration after completion of the cultivation and the specific activity of the cells were lower than those obtained in a breaker-scale tank, the cultivation tank could not be smoothly scaled up, and the expression of the nitrile hydratase activity was insufficient.
While the causes for such phenomena are not clear, increased shear force of stirring blades due to the scaling up of the cultivation tank required to thereby maintain the above oxygen feed rate during cultivation may not be considered to have some influence on the growth of the bacteria directly or indirectly. In any case, the bacteria of the present invention cannot be cultivated on a commercial scale unless these problems are settled.